The high degree of compound selectivity made possible by combining liquid chromatography with molecular detector methods which provide structural information has been recognized as extremely valuable for the identification of various components of complex mixtures. Particularly, liquid chromatographs (LC), and especially high-performance liquid chromatographs (HPLC), have proven to be excellent means for separating a mixture and determining the individual constituents, either quantitatively or volumetrically. However, LC and HPLC devices have the disadvantage that they do not satisfactorily identify the separated constituents.
On the other hand, the mass spectrometer (MS) is extremely capable and sensitive in identifying single components, while having considerable difficulty in identifying a mixture. Consequently, hybrid techniques which combine chromatography with molecular methods such as mass spectrometry and fourier transform infrared spectrometry have been developed and are used extensively for component analysis in complex mixtures.
The high scan speed and sensitivity of fourier transform infrared (FTIR) spectroscopy have enabled the recording of infrared spectra of individual components of a mixture which have been separated by chromatographic techniques. Coupling of FTIR equipment has been successfully accomplished for gas chromatography (GC), however, many compounds and mixtures are not sufficiently volatile for GC separation. Moreover, the sensitivity of a combination GC/FTIR mechanism is reduced for less volatile compounds, making this combination unacceptable. Particularly, the less volatile and/or more polar compounds must usually be separated by HPLC.
Interfacing of LC mechanisms with FTIR devices has not been substantially successful heretofore due to the infrared absorption of the mobile phase of the HPLC eluent. Generally, solvents which are good mobile phases for LC and HPLC applications are also usually strong infrared absorbers. To address this problem, two general types of systems have been developed; (1) flow cells which take advantage of some mobile phases which have large IR windows; and (2) elimination of the mobile phase prior to deposition of the eluate on an appropriate substrate. Each of these approaches, however, have their own problems in achieving a reliable and universal interface arrangement.
For example, all solvents absorb some infrared radiation, and the degree of such absorption defines the maximum path length which a flow cell can have which will allow identifiable spectra to be obtained. Additionally, mobile phases having large IR windows are generally of low polarity and are used only for normal-phase HPLC. The shorter path lengths which must be used to minimize interference resulting from mobile phase absorption similarly limit the volume of the flow cell, thereby limiting the concentration of the analyte being measured at any one instant, and compromising the accuracy of the process overall. The major challenge of interfacing normal-phase and reverse-phase HPLC to IR techniques is the incompatibility of typical solvents to identification of unknown constituents by IR technology. Consequently, water and other typical mobile phases used in LC separations are best eliminated prior to measuring the IR spectrum of a component.
A variety of methods and devices have been directed toward eliminating solvents prior to FTIR procedures, including flowing effluent from a capillary HPLC column into a stainless steel wire net designed to eliminate the solvent as a result of a heated gas flow. Particularly, the sample material was to be suspended between the metal meshing, and the deposits were then analyzed. Griffiths et al. developed a system wherein the HPLC effluent is deposited on an IR transparent substrate as warm nitrogen induces solvent evaporation prior to IR analysis. An interface where deposition of the sample material was to be continuous was developed by Gagel and Biemann, where effluent from a microbore HPLC was continuously sprayed on a rotating disk as warm nitrogen was passed across the disk to evaporate the solvent. In that procedure, however, the FTIR spectra were measured off-line by fastening the collection device to a reflectance accessory.
A solvent removal interface developed by Kalasinsky for reverse phase HPLC contemplated the elimination of water by employing a particular chemical (2,2'-dimethoxypropane) to convert the water to methanol and acetone for deposition on a KCl substrate. Such conversion requires specific matching of chemicals and collection substrates, and does not truly remove the solvent but merely converts it to other substances which can independently add interference to analysis results. Browner and coworkers developed a monodisperse aerosol generator interface for combining LC and FTIR spectrometry, known as the MAGIC interface. With this interface, mobile phase elimination was to be accomplished at room temperature, wherein effluent from an HPLC enters the interface through a 25 micrometer diameter orifice to form a liquid jet. The jet is dispersed by a Helium (He) stream to create a fine aerosol which is directed from a desolvation chamber into first and second momentum separators. In the first momentum separator, evaporated solvent and Helium are removed by vacuum pumps, and the nonvolatile analyte continues into the second momentum separator where any residual volatile material is to be removed. The nonvolatile analyte is then deposited on a KBr (potassium bromide) window which is removed and placed in a beam condenser for IR analysis. Because the solvent is eliminated prior to deposition on the substrate, the isolated analyte can be deposited on a variety of substrates for various IR detection methods.
In U.S. Pat. Nos. 4,814,612 and 4,883,958, M. L. Vestal, et at. described similar apparatuses and methods for coupling LC and solid phase detectors, including the use of thermospray vaporizers which vaporize most of the solvent prior to introduction to a desolvation chamber. The device set forth in the '958 patent further contemplates passing the vaporized solvent and added carrier gas through one or more solvent removal chambers, which can remove solvent by condensation or diffusion through a membrane to a counterflowing gas stream. This device may further include a momentum separator to concentrate particles relative to the remaining solvent vapor and carrier gas, and teaches the direction of a particle beam for impact with a cryogenically cooled deposition surface. In the Vestal '612 patent, a moving belt is provided for receiving the particle beam, and a temperature transducer is positioned adjacent the belt to maintain the belt at a temperature where no significant amount of the particle sample will be vaporized, yet warm enough that residual liquid solvent is vaporized efficiently in a stream of counterflowing gas which passes over the belt.
The Vestee Universal Interface incorporates many of the features described in the Vestal patents mentioned above, and is available in the industry from Vestec Corporation of Houston, Tex.
An apparatus for combining LC technology with mass spectrometry is described in U.S. Pat. No. 4,980,057, which issued to S. B. Dorn, et al. The Dorn, et al. device includes a nebulizer which volatilizes the LC eluate to form an aerosol which passes through a desolvation chamber. The nebulizer introduces an inert gas which helps vaporize the solvent and carries the aerosol to a momentum separator which accelerates the particles to sonic velocities. The momentum separator includes three vacuum pumping stages, wherein the first two stages are defined by conical skimmer nozzles, and the third chamber includes a long inlet tube which provides the vacuum pumping restriction. The resulting particle beam is provided to the MS ion source for analysis.
Consequently, while a great number of investigations and techniques have been attempted heretofore, LC/FTIR interfaces have provided only limited success in providing interpretable IR spectra from normal-phase and reverse-phase separations, due to inadequate solvent elimination and/or limited applicability to IR analysis.